Method for stimulating T lymphocytes with IL-2

ABSTRACT

The present invention relates to novel therapies for treating autoimmune and inflammatory diseases. More specifically, the present invention relates to a use of low dose interleukin-2 for the treatment of type I diabetes and other autoimmune and/or inflammatory diseases.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational PCT application PCT/EP2012/054174, filed Mar. 9, 2012,which claims priority from European Patent Application 11305269.0 filedMar. 11, 2011 and U.S. Provisional Application 61/451,663 filed Mar. 11,2011, each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel IL-2-based therapies of humandiseases. More specifically, the present invention relates to low dosesIL-2 therapies of autoimmune, immune-related, or inflammatory diseasesincluding inflammation in human subjects.

BACKGROUND OF THE INVENTION

Interleukin-2 (IL-2) was identified almost 30 years ago [1] andoriginally called T cell growth factor because of its ability tostimulate T lymphocytes in vitro. It is a protein with a reportedmolecular weight of between approx. 13 kda and 17 kda [2] and anisoelectric point of approx. 6 to 8.7.

IL-2 has been used in the clinic for boosting effector immune responsesin cancers and infectious diseases [3, 4]. It is now authorized for usein human for the treatment of cancer.

In one of its registered indications, adjunct treatment of renal cellcarcinoma (RCC), less than 10% of the patients respond to treatment.This limited efficacy of IL-2 is now partly explained by the recentdiscovery that IL-2 also plays a major role in the peripheral survivaland suppressive function of regulatory T cells (Tregs) [5, 6], which areknown to supress anti-tumor effector responses.

In fact, IL-2/IL-2 receptor (IL-2R) signalling is important duringimmune responses of both effector T cells (Teff) and Treg. On the onehand, extensive IL-2R signalling is necessary for the development ofterminally differential short-lived Teff cells that exhibit enhancedfunctional activity, and for eliciting proper T cell memory [7]. On theother hand, IL-2/IL-2R signalling is essential for Treg development andhomeostasis as shown by the fact IL-2 knock-out mice lack Tregs.Noteworthy, IL-2 or IL-2R deficient mice are able to mount effectorimmune responses, as notably attested by their development of severeT-cell mediated auto-immune diseases (AID).

These different consequences of IL-2 signalling abnormalities are nowexplained by the fact that both quantitative and qualitative differencesin IL-2/IL-2R signalling regulate Treg and Teff. Tregs appear to requirelow IL-2/IL-2R signalling threshold to support their development andperipheral homeostasis [6]. IL-2 administration has been shown to leadto marked expansion and activation of Tregs in mice and humans [3, 4,8].

Nowadays, IL-2 continues to be utilized exclusively for cancerimmunotherapy, and has not been investigated in human auto-immunediseases or, more generally, in human diseases caused by an undesirableimmune response. This is because of the perceived and expected risksassociated with such treatment. Indeed, the capacity of IL-2 tostimulate Teffs carries the risk of activating the very effector T cellsthat mediate the disease and therefore to aggravate the disease.

SUMMARY OF INVENTION

An object of the invention relates to a method for reducing orpreventing an undesirable immune response in a human subject, the methodcomprising administering to said subject an amount of interleukin-2(IL-2) efficient to stimulate regulatory T lymphocytes (Tregs) withoutsubstantially inducing effector T lymphocytes (Teffs).

A further object of the invention relates to a method for reducing orpreventing an undesirable immune response in a human subject, the methodcomprising administering to said subject an amount of interleukin-2(IL-2) efficient to stimulate regulatory T lymphocytes (Tregs) withoutsubstantially inducing IL-2-associated side effects.

A further object of the invention relates to a method for reducing orpreventing an undesirable immune response in a human subject, the methodcomprising administering to said subject an amount of interleukin-2(IL-2) efficient to stimulate regulatory T lymphocytes (Tregs) withoutsubstantially inducing effector T lymphocytes (Teffs) andIL-2-associated side effects.

A further object of the invention relates to a method for reducing orpreventing an undesirable immune response in a human subject, the methodcomprising administering to said subject an amount of interleukin-2(IL-2) efficient to shift the balance (or ratio) between regulatory Tlymphocytes (Tregs) and effector T lymphocytes (Teffs) (Treg/Teffbalance) towards Tregs in said subject.

A further object of the invention relates to a method for reducing orpreventing an undesirable immune response in a human subject, the methodcomprising administering to said subject an amount of interleukin-2(IL-2) efficient to increase the balance (or ratio) between regulatory Tlymphocytes (Tregs) and effector T lymphocytes (Teffs) in said subject.

The present invention proposes novel IL-2-based therapies. The inventionshows, for the first time, that IL-2 can be used as a novel class ofimmuno-regulatory and anti-inflammatory drugs acting by specific Tregexpansion/activation.

A further object of the invention relates to a method for treating anautoimmune, immune-related or inflammatory disorder in a human subject,the method comprising administering to said subject an amount ofinterleukin-2 (IL-2) efficient to shift the balance between regulatory Tlymphocytes (Tregs) and effector T lymphocytes (Teffs) (Treg/Teffbalance) towards Tregs in said subject.

A further object of the invention relates to a method for treating anautoimmune, immune-related or inflammatory disorder in a human subject,the method comprising administering to said subject an amount ofinterleukin-2 (IL-2) efficient to stimulate regulatory T lymphocytes(Tregs) without substantially inducing effector T lymphocytes (Teffs),and/or to decrease inflammation.

The invention may be used for curative or preventive treatment of suchdisorders. More particularly, the methods of the invention preventoccurrence or development of an autoimmune, immune-related orinflammatory disorder in a subject.

IL-2 is typically administered repeatedly.

Interleukin-2 for use in treating an autoimmune, an immune-related or aninflammatory disorder according to claim 1, wherein it is to beadministered at a dose of about 0.05 to about 2 millions internationalunit (MIU)/m²/day, preferably about 0.1 or 0.2 to about 1 MUI/m²/day orat a dose of less than about 3.5 MIU/day.

In a preferred embodiment, it is administered at a dose of about 3MIU/day or at a dose of less than about 2 MIU/day, preferably at a doseof between about 0.1 MIU and about 2 MIU/day, preferably of betweenabout 0.3 MIU and about 1 MIU/day.

The treatment may preferably comprise at least a first course whereininterleukin-2 is administered once per day during at least 3 consecutivedays, preferably during 3 to 7, still preferably 4 to 5 consecutivedays. Preferably it is followed by a maintenance dose after two to fourweeks. The maintenance dose can be administered once a week, or once ortwice a month.

In a preferred embodiment, the treatment comprises at least a firstcourse wherein a dose of about 0.2 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.2 MUI/m² after one to three weeks, whichmaintenance dose can be repeated every one to three weeks. In apreferred aspect, the subject is an adult that is administered withabout 0.3 MUI as the daily dose of 0.2 MUI/m².

In another preferred embodiment, the treatment comprises at least afirst course wherein a dose of about 0.6 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.6 MUI/m² after two to 4 weeks, whichmaintenance dose can be repeated every two to four weeks. In a preferredaspect, the subject is an adult that is administered with about 1 MUI asthe daily dose of 0.6 MUI/m².

In still another preferred embodiment, the treatment comprises at leasta first course wherein a dose of about 1.8 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 1.8 MUI/m² after about one to two months,which maintenance dose can be repeated every one to two months. In apreferred aspect, the subject is an adult that is administered withabout 3 MUI as the daily dose of 1.8 MUI/m².

Generally speaking, it is preferred that IL-2 is administered at a doseof D/10 to 20×D, preferably D/5 to 10×D, wherein D is the minimal dosetriggering up-regulation of expression of CD25 in Treg, without inducingexpansion of Treg. Preferably the up-regulation of expression of CD25 isat least 33%, preferably at least 50%.

The invention stimulates regulatory T lymphocytes (Tregs) withoutsubstantially inducing effector T lymphocytes (Teffs) in a humansubject. The methods of the invention make it possible to increase theTreg/Teff balance or to increase potent suppressive Treg cell populationin said subject.

IL-2 is further administered for reducing or preventing inflammation ina human subject.

The invention may be used for the treatment or prevention of anycondition associated to or caused by an undesirable immune response. Itis particularly suited for treating inflammatory, immune-related orautoimmune diseases, including without limitation HCV-relatedvasculitis, uveitis, myositis, type I diabetes, systemic lupuserythematous, systemic vasculitis, psoriasis, allergy, asthma, Crohn'sdisease, Multiple Sclerosis, Rheumatoid Arthritis, atherosclerosis,autoimmune thyroid disease, neuro-degenerative diseases, Alzheimerdisease, graft-versus-host disease, spontaneous abortion and allograftrejection.

Another subject of the invention is a method for determining whether aIL-2 regimen or dose has to be modified in a patient with an autoimmune,an immune-related or an inflammatory disorder, treated with IL-2, whichmethod comprises monitoring the quantity of Tregs and/or CD25 expressionlevel in Tregs, wherein a quantity of Tregs and/or CD25 expression levelin Tregs inferior to a control value, means that the dose of IL-2 is tobe increased.

LEGEND TO THE FIGURES

FIG. 1: Effects of low dose IL-2 on biological markers of HCV relatedautoimmune vasculitis

Time course changes in HCV viral load (panel A), cryoglobulin (panel B)and C4 complement serum levels (panel C) are shown. Data are expressedas mean±standard errors (n=10, *: P<0.05, **P<0.01, ***: P<0.001).

FIG. 2: Effects of low dose IL-2 on lymphocyte sub populations

Time course changes of percentages of CD4⁺CD25^(hi)CD127⁻Foxp3⁺ in CD4⁺T cells and of CD8⁺CD25⁺Foxp3⁺ in CD8⁺ T cells (panel A), globalTreg/Teff ratio (panel B), absolute numbers of CD19⁺ total B cells andMarginal zone B cells (panel C) and of NK cells and CD56^(bright) NKcells (panel D) are shown. Data are expressed as mean±standard errors(n=10,*: P<0.05, **P<0.01, ***: P<0.001).

FIG. 3. Phenotypic characterization of CD4⁺ Treg by Flow cytometry.Representative lymphocyte gate for identification of the CD3⁺CD4⁺ Tsubset is shown. Within the CD3⁺ CD4⁺ T cells, Tregs were identified asCD25^(high)CD127⁻FoxP3⁺ cells.

FIG. 4. Suppressive activity of Treg cells in HCV related autoimmunevasculitis patients under IL-2 treatment

FACS purified Treg were assayed for their capacity to suppressautologous effector T cells proliferation at different ratios (1/1 to1/8) under allogeneic stimulation. Results are expressed in cpm. Theexperiment shown is representative of 4.

FIG. 5. Phenotypic characterization of CD8⁺ Treg by Flow cytometry.Representative lymphocyte gates for the identification of CD3⁺CD8⁺ Tregare shown. Within CD3⁺CD8⁺ T cells, Tregs were identified as CD25⁺FoxP3⁺cells.

FIG. 6. CD19⁺ B cells subsets characterization by flow cytometry

B cell subset populations were defined as naïve (IgD⁺CD27⁻), memory(IgD^(−CD)27⁺), marginal zone(IgD⁺CD27⁺).

FIG. 7: Low dose IL-2 induces a global decrease of inflammation revealedby transcriptome analyses of PBMCs

Hierarchical clustering before and after IL-2 treatment (panel A)highlights gene downregulations affecting mainly B cell related genesand proinflammatory genes. Data mining identified the NFKB pathway ascentral in the decreased proinflammatory response (panel B).Down-regulated genes in IL-2 treated patients are symbolized in redboxes. Genes present in the transcriptional signature (panel A) areschematically represented in filled round-boxes. Post-transcriptionalactivation between 2 gene products (e.g. by phosphorylation, clivageetc. . . . ) is representated with a sharp arrow and a filled arrowstands for a direct transcriptional activation. Dotted arrows representindirect signalling.

The table (panel C) shows the results of an ICA of these data. Number ofup- or down-regulated signatures in the IL-2-treated group that havesignificant enrichment for GO terms and KEGG pathways related toinflammation, immune response, and autoimmune (type I diabetes mellitus,systemic lupus erythematosus, autoimmune thyroid disease),transplantation (graft-versus-host disease and allograft rejection) orinflammatory infection-related pathologies (Chagas disease,Leishmaniasis, Helicobacter pylori infection, Malaria, Amoebiasis,Shigellosis, all characterized by a high degree of inflammation) areshown. As controls, the same number of randomly-picked GO terms wastested, together with cell-cycle related GO terms and controlpathologies. For each GO term or KEGG pathway, the Khi2 test p-valueindicates a possible enrichment bias for up- or down-regulatedsignatures as compared to the overall up- (2527) or down- (3429)regulated signatures.

FIG. 8: Low dose IL-2 induces Treg in Type I diabetes patients.

FIG. 8 shows the Treg/T_(CD4) ratio in patients administered with 0.33,1 or 3 MUI/day IL-2 (Proleukin®) during 5 days. The peak Treg increaseis relatively similar at the different doses, but the duration of theeffect is dose-dependent, and provides information to define theschedule of the maintenance treatment (i.e. the lower the dose, theshorter the delay between maintenance injections).

FIG. 9: Low dose IL-2 did not decrease C-peptide production.

FIG. 9A shows the Treg/T_(CD4) ratio in a patient administered with IL-2(Proleukin®) during 5 days. FIG. 9B shows the concentration of serumC-peptide before and after administration of IL-2, in the same patient.There is an increase in Treg during treatment, which correlates with anincrease in C peptide production at 2 months.

DETAILED DESCRIPTION OF THE INVENTION

The present invention unexpectedly shows, for the first time, in vivoexpansion of very potent supressive Tregs without expansion of Teff noroccurrence of adverse events through low-dose IL-2 immunotherapy inhuman autoimmune patients.

IL-2 is utilized exclusively for cancer immunotherapy, and has not beeninvestigated alone in human auto-immune diseases in humans. Indeed, thecapacity of IL-2 to stimulate Teffs carries the risk of activating thevery effector T cells that mediate auto-immunity. Here, we showbiological evidence that IL-2 can be used under conditions that induceTregs without inducing Teffs, concordant with clinical evidence showingthat no immune activation-related adverse events were observed. Inaccordance with this invention, IL-2 clearly tips the Treg/Teff balancein favor of Tregs, as also supported by the globaly reduced inflammatorycontext during IL-2 therapy.

In first trials in HCV-related patients, the inventors showed thatlow-dose IL-2 is well tolerated, induces a dramatic and selectiveincrease in Treg cells, and leads to clinical improvement in 80% of thepatients.

With a 5-day 1.5 M IU/day course treatment, a significant increase ofTreg was observed in all patients (a 2 fold increase), with no sideeffects. After additional 5-day courses of 3 M IU/day, a furtherincrease of Tregs was noted.

Our results show the treatment was well tolerated, with no significantchanges in granulocytes, red blood cells, or liver enzymes. Furthermore,at the dosage of 1.5 MIU/day, no side effects were noted such asasthenia, transient local reactions at injection sites, flu-likesyndrome, myalgia or hypertension.

Importantly, during the entire treatment and follow-up, there were nobiological or clinical signs indicating activation of pathogenic Tcells.

A course of 1.5 M IU/day for 5 days represents so far the lowest IL-2dose with proven efficacy and safety for Tregs induction purposes inhumans.

In addition, the present invention revealed, for the first time, amarked anti-inflamatory activity of IL-2. Our unsupervised transcriptomeanalyses showed a clear down-regulation of signatures/pathwaysassociated with many auto-immune and inflammatory diseases, as well asimmune-related diseases such as graft versus host disease and allograftrejection. With this study showing that it is possible to use IL-2 tostimulate Treg without stimulating Teff in humans, the inventors proposethat low dose IL-2 treatments will profoundly change the preventive andtherapeutic paradigms for all these diseases.

The inventors have proceeded further with testing low-dose IL-2 inanother autoimmune disease, namely type I diabetes, thereby confirmingthe interest of low-dose IL-2 in treating autoimmune, immune-related orinflammatory disorders.

The present invention therefore provides new therapeutic approaches fortreating autoimmune, immune-related or inflammatory disorders usingIL-2. The invention discloses that IL-2, at low doses, can effectivelyactivate or expand suppressive Treg cell population in subjects havingautoimmune, immune-related or inflammatory disorders, withoutsubstantially activating effector T cells.

The subject is any human patient, regardless of age or gender. In aparticular embodiment, the patient may be a child, or an adolescent.

Interleukin-2 (IL-2)

Within the context of this invention, the term “IL-2” designates anysource of IL-2, including mammalian sources such as e.g., human, mouse,rat, primate, and pig, and may be native or obtained by recombinant orsynthetic techniques, including recombinant IL-2 polypeptides producedby microbial hosts. IL-2 may be or comprise the native polypeptidesequence, or can be an active variant of the native IL-2 polypeptide.Preferably the IL-2 polypeptide or active variant is derived from ahuman source, and includes recombinant human IL-2, particularlyrecombinant human IL-2 produced by microbial hosts.

Active variants of IL-2 have been disclosed in the literature. Variantsof the native IL-2 can be fragments, analogues, and derivatives thereof.By “fragment” is intended a polypeptide comprising only a part of theintact polypeptide sequence. An “analogue” designates a polypeptidecomprising the native polypeptide sequence with one or more amino acidsubstitutions, insertions, or deletions. Muteins and pseudopeptides arespecific examples of analogues. “Derivatives” include any modifiednative IL-2 polypeptide or fragment or analogue thereof, such asglycosylated, phosphorylated, fused to another polypeptide or molecule,polymerized, etc., or through chemical or enzymatic modification oraddition to improve the properties of IL-2 (e.g., stability,specificity, etc.). Active variants of a reference IL-2 polypeptidegenerally have at least 75%, preferably at least 85%, more preferably atleast 90% amino acid sequence identity to the amino acid sequence of thereference IL-2 polypeptide.

Methods for determining whether a variant IL-2 polypeptide is active areavailable in the art and are specifically described in the presentinvention. An active variant is, most preferably, a variant thatactivates Tregs.

Examples of IL-2 variants are disclosed, for instance, in EP109748,EP136489, U.S. Pat. No. 4,752,585; EP200280, or EP118,617.

Preferably we use a recombinant IL-2, i.e., an IL-2 that has beenprepared by recombinant DNA techniques [9]. The host organism used toexpress a recombinant DNA encoding IL-2 may be prokaryotic (a bacteriumsuch as E. coli) or eukaryotic (e.g., a yeast, fungus, plant ormammalian cell). Processes for producing IL-2 have been described e.g.,in U.S. Pat. No. 4,656,132; U.S. Pat. No. 4,748,234; U.S. Pat. No.4,530,787; or U.S. Pat. No. 4,748,234, incorporated therein byreference.

In a preferred embodiment, the invention uses an IL-2 of human origin,or an active variant thereof, more preferably produced recombinantly. Anucleotide and an amino acid sequence of human IL-2 are disclosed, forinstance, in Genbank ref 3558 or P60568, respectively. The inventionmore preferably uses a human IL-2.

IL-2 for use in the present invention shall be in essentially pure form,e.g., at a purity of 95% or more, further preferably 96, 97, 98 or 99%pure.

For use in the present invention, IL-2 is typically not combined orco-administered with a Teff suppressive agent. However, although notpreferred or required, drug combinations may be contemplated.

IL-2 may be used in monomeric or multimeric form.

IL-2 is commercially available, including for pharmaceutical uses, andit is authorized for use in human patients. Suitable commercial formsinclude, e.g.,

-   -   Proleukin®, a recombinant, human IL-2 composition,    -   Aldesleukin®, an unglycosylated des-alanyl-1, serine-125 human        interleukin-2 produced in E. coli.    -   Roncoleukin®, recombinant human IL-2 produced in yeast.

Interleukin-2 may be used alone or in combination with any othertherapeutically active agent. In a preferred embodiment, when IL-2 isnot administered in combination with rapamycine when used in preventingor treating type I diabetes.

Regulatory T Cells

Regulatory T cells are T lymphocytes having immunosuppressive activity.Natural Tregs are characterized as CD4+CD25+Foxp3+ cells. Humans andmice presenting a genetic deficit in Tregs develop multiple T-cellmediated organ-specific autoimmune diseases. A Treg quantitative orqualitative defect has been described in many human autoimmune diseases,including systemic lupus erythematosis (SLE), Type 1 Diabetes, MultipleSclerosis, uveitis and myositis. Conversely, addition/restoration ofTreg induces clinical improvements in most animal models of thesediseases.

Tregs also play a major role in the control of inflammatory diseases,although their mode of action in such disease is not well understood. Infact, in most inflammatory diseases, Treg depletion exacerbates diseasewhile Treg addition decreases it. This is for example shown in thecontext of atherosclerosis. Although this disease is not primarily aninflammatory disease, its development involves an inflammatorycomponent/loop. In apolipoprotein E (ApoE) deficient mice thatspontaneously develop atherosclerosis, Treg depletion significantlyaggravated the plaque formation, while injection of polyclonal Tregssignificantly improved the disease.

Most Tregs are CD4+ cells, although there also exists a rare populationof CD8+ Foxp3+ T lymphocytes with a suppressive activity.

Effector T Cells

Within the context of this application, “effector T cells” (or “Teff”)designates conventional T lymphocytes other than Tregs (sometimes alsoreferred to as Tconv in the literature), which express one or more Tcell receptor (TCR) and perform effector functions (e.g., cytotoxicactivity, cytokine secretion, anti-self recognition, etc). Majorpopulations of human Teff according to this invention include CD4+ Thelper lymphocytes (e.g., Th0, Th1, Th17) and CD4+ or CD8+ cytotoxic Tlymphocytes, and they can be specific for self or non-self antigens.

In the particular situation of an autoimmune disease, the Teff cellsinclude the T cell population responsible for or involved in thedisease. For instance, such population of T cells include T lymphocyteswhich recognize a self antigen such as a thyroid antigen, a jointantigen, a β-Langherans islet antigen, etc. In a GVHD disease, the Teffcells include T lymphocytes from the graft.

Selective Amplification of Tregs

Within the context of this invention, a stimulation (or induction oractivation or amplification) of Treg designates any increase inproportion of Treg cells relative to Teffs, in number or in activity astested by suppressive assays or by expression of molecules that reflectthe activity of the Tregs such as CD25, the alpha-chain of the IL-2receptor, in a patient. The augmentation in proportion is preferably byat least 20% as compared to the level prior to treatment, morepreferably at least 40%, even more preferably at least 60%. In aparticular and preferred embodiment, the stimulation designates a shiftin the Treg/Teff balance towards Tregs, or an increase in the Treg/Teffratio.

An essential aspect of the invention lies indeed in the ability, invivo, in human patients having an autoimmune, immune-related orinflammatory disorder, to stimulate Tregs without substantially inducingTeff.

The induction (or activation or expansion) of Tregs can be measured asdisclosed in the examples, e.g., by measuring the number of Tregs (e.g.,based on the expression of CD25, FoxP3 . . . ) and/or the activity ofTregs in samples from the treated subject. The absence of substantialinduction (or activation or expansion) of Teff can also be measured asdisclosed in the examples, e.g., by measuring the number of Teff and/orthe activity of Teff in samples from the treated subject. Prerefably,the absence of substantial induction indicates the target Teff cellpopulation does not acquire markers of activation such as CD25, CD69,and/or HLA-DR, or as assessed by whole transcriptome analyses. Detailedmethods for detecting, measuring and quantifying Treg and Teff cells arewell known per se in the art and some are disclosed in the examples.

Stimulation in Tregs may be measured by an increase in Treg counts inthe patient, typically by 10% at least, or by an increase in activationmarkers such as the intensity of CD25 expression. The absence of Teffinduction typically designates that the Teff cell population has notincreased by more than 10% in said subject as a result of treatment.

The stimulation of Treg and absence of substantial induction of Teff ispreferably assessed by a measure of the ratio or the balance Treg/Teffin the treated subject. This balance is calculated e.g., based on thenumber of Tregs and the number of Teff in a sample from the subject. Asillustrated in the examples, such a balance typically increases by atleast 20% in the treated patients, more preferably by at least 30%, 40%or 60%.

The baseline percentage of Tregs in human subjects, i.e., the ratio ofTregs/CD4+Teffs, is approx. 4.6±0.6% (the baseline % in patients withautoimmune disease may be much lower, as shown in Example 1, wherepatients with HCV-induced autoimmune vasculitis had a baselinepercentage level of 3.6%±0.23 only, although these numbers may vary fromone laboratory to another, based on technical aspects of themeasurement).

The results presented in this application show that, after 1 course oftreatment with 1.5 MIU/day, a 2-fold (100%) increase in the baseline %of Tregs is obtained, which may be further amplified upon additionaltreatment. Depending on the protocol, increases above 300% have beenobtained. A similar 2-fold increase after 1 5-Day course has beenobserved in patient with T1D, whatever the dose used (0.33; 1 and 3MIU/day).

In a preferred embodiment, the method allows an increase by 20%, 30%,40%, 50%, 75%, 100% or more of the Treg/Teff ratio in a subject.

Furthermore, the invention shows an increase not only in CD4+ Tregs, butalso in a rare population of Tregs which are CD8+.

In a particular embodiment, the invention increases circulatingCD4⁺CD25^(hi)CD127⁻Foxp3⁺ Tregs.

In another particular embodiment, the invention increases circulatingCD8⁺CD25^(hi)Foxp3⁺ Tregs.

Another important aspect is that the amplified Treg cell population ishighy suppressive. Indeed, the results presented show that, upontreatment according to the invention, Tregs are activated having apotent suppressive activity towards Teff cells. In our study, asubstantial suppressive activity (>75%) could be detected at Treg/Teffratio of 1/8 in a classical suppressive assay in which Treg from nontreated normal individual gives such a suppression at ratio of 1/2 to1/4.

The invention therefore allows a substantial increase in Tregs in humansubjects having autoimmune diseases, without substantial alteration oractivation of effector T lymphocytes. In a preferred embodiment, themethod of the invention is a method that increases by at least 30% theCD25⁺Foxp3⁺ Tregs in a subject and that causes less than 5% increase intarget Teffs in said subject.

Also, the invention unexpectedly shows that, at preferred administrationdoses, the IL-2-based therapies of the invention essentially do notinduce arterial hypertension; headaches, nausea, arthralgia, myalgia, orFlu-like syndrome, nor many other IL-2 known side effects as describedin the IL-2 Summary of Product Charasteristics of the FDA). It istherefore possible to use IL-2 therapy in human subjects havingimmune-related disorders, without substantially activating Teff cellsand without substantially inducing IL-2-associated side effects, whilevery substantially inducing Tregs and anti-inflammatory effect.

IL-2 Dosage

For use in the present invention, IL-2 is administered at a dosage whicheffectively activates Tregs without substantially activating Teffs. Theconsequence is a dramatic increase in the Treg/Teff balance in thesubject.

The effective dosage can be adjusted by the practitioner, based oninformation contained in the present application. In particular, withthe knowledge of the present invention that, in patients with autoimmunedisease, IL-2 may be administered under conditions which do activateTregs and which essentially do not activate Teff, the skilled person maybe able to adjust dosages to each patient and condition.

Typically IL-2 is administered at a dose of about 0.05 to about 2MIU/m²/day, preferably 0.2 to about 1 MIU/m²/day.

The amount of IL-2 to administer thus preferably depends on the bodysurface area of the subject. The body surface area (BSA) is the measuredor calculated surface of a human body.

Various calculations have been published to arrive at the BSA withoutdirect measurement:

The Dubois & Dubois formula [18] is commonly used in adults:

${{BSA}\left( m^{2} \right)} = {{0.007184 \times {weight}\mspace{14mu}({kg})^{0.425} \times {height}\mspace{14mu}({cm})^{0.725}} = \frac{{weight}\mspace{14mu}({kg})^{0.425} \times {height}\mspace{14mu}({cm})^{0.725}}{139.2}}$

Another commonly used formula is the Mosteller formula [19] adopted foruse by the Pharmacy and Therapeutics Committee of the Cross CancerInstitute, Edmonton, Alberta, Canada:

${{BSA}\left( m^{2} \right)} = {\left. \sqrt{}\begin{matrix}{{weight}\mspace{11mu}({kg}) \times {height}\mspace{11mu}({cm})} \\3600\end{matrix} \right. = \begin{matrix}{{weight}\mspace{11mu}({kg})^{0.5} \times {height}\mspace{11mu}({cm})^{0.5}} \\60\end{matrix}}$

It is more particularly used in children.

Average BSA is generally taken to be 1.73 m² for an adult.

Average BSA values Neonate (Newborn) 0.25 m² Child 2 years  0.5 m² Child9 years 1.07 m² Child 10 years 1.14 m² Child 12-13 years 1.33 m² For men 1.9 m² For women  1.6 m²

Typically, the dosage according to the invention is below 3.5 MillionIU/day/patient, more preferably below 3.0 Million IU/day/patient, evenmore preferably below 2.5 Million IU/day/patient, further preferablybelow 2.0 Million IU/day/patient.

The treatment is typically repeated, i.e., the above low doses IL-2 areadministered several times to a subject, to progressively achieve themost substantial benefit. The dose and schedule of administration varyaccording to the preventive or therapeutic aim of the treatment, as wellas to the disease to be treated/prevented. Treatment effect can bemonitored by Treg measurements and dose and administration scheduleadjusted accordingly.

Examplary dosages are between 0.1 to 3 MIU, preferably 0.1 to 1.5 MIU,still preferably 0.25 to 1 MUI. Preferred dosages are:

-   -   3.0 M IU/day/patient,    -   2.5 M IU/day/patient,    -   2.0 M IU/day/patient,    -   1.5 M IU/day/patient,    -   1.0 M IU/day/patient,    -   0.5 M IU/day/patient,    -   0.3 MIU/day/patient    -   0.1 M IU/day patient,    -   0.05 M IU/day patient,    -   0.02 M IU/day patient, or    -   0.01 M IU/day patient.

These dosages may be combined, depending on the subject and evolution ofthe disease.

Treatment may be provided as courses of several days, e.g., 1-7 days ofdaily administration, preferably between 3 to 5 days. Such treatmentcourses may be reproduced at different time periods, interrupted byperiods with no treatment. In a preventive setting, IL-2 may beadministered at the above dosage in single shots, at different timeintervals, e.g., once a week over long periods of time. Differentprotocols may be adjusted depending on the patient and disease.

Maintenance dosage can be administered from two to eight weeks after theinitiating cycle is completed. Preferably the maintenance dose is thesame as the initiating dose.

Determination of Dosage

Generally speaking, IL-2 may be administered at a dose of D/10 to 20×D,preferably D/5 to 10×D, wherein D is the minimal dose triggeringinduction of expression of CD25 in Treg, without inducing expansion ofTreg.

This method for determining the appropriate low-dose of IL-2 isparticularly useful when a route of administration different from thesubcutaneous route is contemplated.

Especially such dosage may be useful in oral, nasal or rectal delivery.

Determination of CD25 levels can be accomplished using anti-CD25antibodies in flow cytometry.

In this regard, lymphocyte-containing samples may be fixed with asuitable fixing agent (e.g. paraformaldehyde, which may be used at 1% inphosphate-buffered saline (PBS)) to permit the subsequent quantificationor qualitative determination of the cell surface marker (e.g. by the useof flow cytometry) as convenient (e.g. following transport from the siteof collection and culture of the lymphocyte-containing sample, to a flowcytometry laboratory). Commercially available Anti-CD25 monoclonalantibodies (mAbs) labeled to different fluorochrome such as Alexa488(Molecular Probes, Oregon, USA) and FITC (Sigma) are available.

Examples of dose and administration schedules are given below.

Treatment of Type 1 Diabetes

Type 1 diabetes (T1D) is due to the auto immune destruction of theinsulin-producing cells in the pancreas. In many patients a defect inthe IL-2/IL-2 receptor activation pathway can be found. Furthermore,IL-2 can prevent the occurrence of type 1 diabetes in mouse models ofT1D (NOD mice) and that IL-2 given very early after the onset ofdiabetes can reverse diabetes.

However, the occurrence of new onset of T1D has been described inpatients with cancer treated with low dose of IL-2. Therefore thetreatment of human T1D patients with IL-2 is not straightforward and noone had yet started such a treatment with IL-2 alone. Example 2 showsthe first results in a double blinded trial in T1D patients administeredwith low dose IL-2 (0.33, 1 or 3 MIU/day) during 5 days.

The inventors thus now disclose a method to 1) prevent and 2) treat T1Dusing low dose of IL-2, especially recent onset T1D.

Interleukin-2 is particularly recommended in a subject at risk ofdeveloping type I diabetes. In that case, the treatment with IL-2 ispreventive of onset of the diabetes in said subject. In the setting ofprevention, patient at risk of T1D (i.e. patients from family with ahistory of T1D or patient with genetic polymorphism of the IL-2/IL-2receptor activation pathway associated with higher frequency of T1D,and/or patients in whom auto-antibodies against antigens such asinsulin, GAD, insulinomaassociated protein 2 (IA2), and tyrosinephosphatase or zinc transporter 8 (ZnT8) (or antibodies that have beenassociated with T1D) have been detected) are treated with low dosesIL-2, thereby preventing the activation of the effector T cells thatwill generate T1D.

In this setting, the treatment uses doses of IL-2 equal or below 3 MIU/day, or even in a preferred embodiment below 1 or 0.5 M IU per day,and the treatment is administered once every two weeks, or preferablyonce every 3 weeks or preferably once every month. Treatment can beadjusted (dose or schedule) based on Treg proportion and function.

In another particular embodiment, a preferred candidate treat shows anunderproduction of IL-2, and a residual production of insulin.

Indeed, in the therapeutic setting, our preferred use is for patientsjust diagnosed with T1D who are known to still have a remaining mass ofinsulin-producing cells in the pancreas. In a particular embodiment,Interleukin 2 in this setting can be given at a dose of 3 million unitsper day for 3 to 7 days and then additional IL-2 courses can be given atleast once a month as described above. Treatment can be adjusted (doseor schedule) based on Treg proportion and function.

In a preferred embodiment, the treatment comprises at least a firstcourse wherein a dose of about 0.2 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.2 MUI/m² after one to three weeks, whichmaintenance dose can be repeated every one to three weeks. In apreferred aspect, the subject is an adult that is administered withabout 0.3 MUI as the daily dose of 0.2 MUI/m².

In another preferred embodiment, the treatment comprises at least afirst course wherein a dose of about 0.6 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.6 MUI/m² after two to 4 weeks, whichmaintenance dose can be repeated every two to four weeks. In a preferredaspect, the subject is an adult that is administered with about 1 MUI asthe daily dose of 0.6 MUI/m².

In still another preferred embodiment, the treatment comprises at leasta first course wherein a dose of about 1.8 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 1.8 MUI/m² after about one to two months,which maintenance dose can be repeated every one to two months. In apreferred aspect, the subject is an adult that is administered withabout 3 MUI as the daily dose of 1.8 MUI/m².

The subcutaneous route is preferred.

Treatment and Prevention of Multiple Sclerosis Relapse

Patient with multiple sclerosis (MS) can be treated with IL-2 to preventor treat relapses. Patient with multiple sclerosis are prone to haverelapse.

In a preferred embodiment, the treatment comprises at least a firstcourse wherein a dose of about 0.2 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.2 MUI/m² after one to three weeks, whichmaintenance dose can be repeated every one to three weeks. In apreferred aspect, the subject is an adult that is administered withabout 0.3 MUI as the daily dose of 0.2 MUI/m².

In another preferred embodiment, the treatment comprises at least afirst course wherein a dose of about 0.6 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.6 MUI/m² after two to 4 weeks, whichmaintenance dose can be repeated every two to four weeks. In a preferredaspect, the subject is an adult that is administered with about 1 MUI asthe daily dose of 0.6 MUI/m².

In still another preferred embodiment, the treatment comprises at leasta first course wherein a dose of about 1.8 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 1.8 MUI/m² after about one to two months,which maintenance dose can be repeated every one to two months. In apreferred aspect, the subject is an adult that is administered withabout 3 MUI as the daily dose of 1.8 MUI/m².

In a particular setting of prevention according to this invention, a MSpatient is treated with low doses IL-2 to prevent relapses. In thissetting, the treatment uses doses of IL-2 equal or below 3 M IU/day, oreven in a preferred embodiment below 1.5 M IU per day, and the treatmentis administered once every two weeks, or preferably once every 3 weeksor preferably once every month. Treatment can be adjusted (dose orschedule) based on Treg proportion and function.

In the therapeutic setting, a MS patient undergoing a relapse can begiven IL-2 at a dose of 3 million units per day for 5 to 7 days and thenadditional IL-2 courses can be given once a month as describe above.Treatment can be adjusted (dose or schedule) based on Treg proportionand function.

Prevention and Treatment of Atherosclerosis

Exemples (non limiting) of uses are 1) for patients with moderateatherosclerosis, i.e. non symptomatic arterial stenosis who can receivemonthly injection of low dose of IL-2 to prevent worsening of thecondition; 2) for patients with aortic aneurysm who can receive monthlyinjection of low dose of IL-2 to prevent progressive increase ofaneurysm size and worsening of the condition; 3) for patients withcoronary or peripheral artery stenosis treated by angioplastywith/without stenting, to reduce inflammation and the risk ofrestenosis, IL-2 at a dose of 3 million units per day for 5 to 7 daysand then additional IL-2 courses once a month; 4) after arterial bypasssurgery to reduce inflammation and risk of restenosis, IL-2 at a dose of3 million units per day for 5 to 7 days and then additional IL-2 coursesonce a month.

In a preferred embodiment, the treatment comprises at least a firstcourse wherein a dose of about 0.2 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.2 MUI/m² after one to three weeks, whichmaintenance dose can be repeated every one to three weeks. In apreferred aspect, the subject is an adult that is administered withabout 0.3 MUI as the daily dose of 0.2 MUI/m².

In another preferred embodiment, the treatment comprises at least afirst course wherein a dose of about 0.6 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 0.6 MUI/m² after two to 4 weeks, whichmaintenance dose can be repeated every two to four weeks. In a preferredaspect, the subject is an adult that is administered with about 1 MUI asthe daily dose of 0.6 MUI/m².

In still another preferred embodiment, the treatment comprises at leasta first course wherein a dose of about 1.8 MUI/m² of interleukin-2 isadministered once a day during at least 3 consecutive days, preferablyduring 3 to 7, still preferably 4 to 5 consecutive days, followed by amaintenance dose of about 1.8 MUI/m² after about one to two months,which maintenance dose can be repeated every one to two months. In apreferred aspect, the subject is an adult that is administered withabout 3 MUI as the daily dose of 1.8 MUI/m².

Prevention of Repeated Spontaneous Abortion

Some couple experience difficulties in reproduction, with recurrentspontaneous abortion which are considered of immunological origin.

In a mouse model of recurrent abortion we have shown that administrationof IL-2 prior to mating allow normal pregnancy outcome.

In female patients with recurrent spontaneous abortion, IL-2 can begiven prior to a programmed conception or implantation of embryosobtained by in vitro fertilisation, to favor fetus implantation. As anexemple, IL-2 courses of 3 M IU per day for 5 days can be given a monthto one week prior to a scheduled mating or implantation.

Prevention of Cell or Organ Rejection

After transplantation of potentially immunogenic allogenic cells ortissues or cell genetically modified with transgene, the administrationof IL-2 according to this invention can be used to reduce the rejectionrate.

Additional examples of Regimen for controlling flare:

-   -   Continuous 24 h infusion for 5 to 7 days at 0.1 to 3.5 or 3 M        IU/day    -   Repeated dosing once daily for 5 to 30 days at daily dosage from        0.1 to 3.5 or 3 M IU/day    -   Repeated dosing once every two days for 15 to 30 days at daily        dosage from 0.1 to 3.5 or 3 M IU/day    -   Repeated dosing once daily for 3 consecutive days in a week        during 2 to 4 consecutive weeks at daily dosage from 0.1 to 3.5        M or 3 IU/day

Examples of Regimen for maintenance and/or prevention:

-   -   Repeated dosing daily for one to seven day in a week every two        to six weeks at daily dosage from 0.1 to 3.5 or 3 M IU/day    -   Repeated dosing once daily for 5 to 30 days every 3 to 6 month        at daily dosage from 0.1 to 3.5 or 3 M IU/day    -   Repeated dosing once daily for 5 to 30 days once a year at daily        dosage from 0.1 to 3.5 or 3 M IU/day.    -   Repeated dosing once daily for 1 to 3 days, every 1 to 3 months,        at daily dosage from 0.1 to 3.5 or 3 M IU/day.    -   Maintenance dosing at 0.01 to 1 M IU/day, 1 day every weeks

The need for administering multiple cycles of a constant IL-2 dosingregimen or multiple cycles of a multi-level IL-2 dosing regimen is atthe discretion of the managing physician and can be assessed bymonitoring Treg cells in subjects undergoing treatment with the methodof the invention.

In a preferred embodiment, in a patient with autoimmune disease, it isbest to reach and/or maintain the Tregs percent level between 5-10%total T cells.

In a preventive setting, it is desirable to reach and/or maintain Tregspercent level between 4.5-7% total T cells.

IL-2 doses may be adapted according to patients' response, i.e. theeffects on Treg percentage and their activation status (CD25).

It is thus described a method for determining whether a IL-2 regimen ordose has to be modified in a patient with an autoimmune, animmune-related or an inflammatory disorder, treated with IL-2, whichmethod comprises monitoring the quantity of Tregs and/or CD25 expressionlevel in Tregs.

A quantity of Tregs and/or CD25 expression level in Tregs inferior tothe control value, means that the dose of IL-2 is to be increased. Thecontrol value is generally the baseline quantity of Tregs and/or CD25expression level in the patient, before any treatment.

In a particular embodiment, such quantification can be conducted whenthe treatment is initiated (e.g. between 3 to 5 days after the firstadministration). If Tregs percentages or CD25 expression levels arebelow a 20% increase compared to baseline, the dose of IL-2 can beincreased (e.g. ×2) and the process repeated until a dose (below 3.5MIU/day) inducing proper Treg response is found.

Preferably this method is also conducted during the maintenance period,which involves quantifying the number of Tregs and/or the expressionlevel of CD25 in Tregs every 2 to 6 months, preferably between 1 to 5days after administration of IL-2. If Tregs percentages or CD25expression levels are below a 20% increase compared to baseline, thedose of IL-2 could be increased (e.g. ×2).

Administration Forms and Routes

Il-2 may be administered using any acceptable method known per se in theart. Thus, for example, IL-2, or the pharmaceutical compositioncomprising IL-2, can be administered by any form of injection, includingintravenous (IV), intramuscular (IM), or transdermal or subcutaneous(SC) injection, or by oral or nasal route as well as by topicaladministration (cream, droplets, etc.). In a particular embodiment ofthe invention, IL-2 is used as a sustained-release formulation, or aformulation that is administered using a sustained release device. Suchdevices are well known in the art, and include, for example, transdermalpatches, and miniature implantable pumps that can provide for drugdelivery over time in a continuous, steady-state fashion at a variety ofdoses to achieve a sustained-release effect. Sublingual or eye dropformulations may also be contemplated.

IL-2 is typically administered in association (e.g., in solution,suspension, or admixture) with a pharmaceutically acceptable vehicle,carrier or excipient. Suitable excipients include any isotonic solution,saline solution, buffered solution, slow release formulation, etc.Liquid, lyophilized, or spray-dried compositions comprising IL-2 orvariants thereof are known in the art and may be prepared as aqueous ornonaqueous solutions or suspensions.

Preferably the pharmaceutical compositions comprise appropriatestabilizing agents, buffering agents, bulking agents, or combinationsthereof to minimize problems associated with loss of protein stabilityand biological activity during preparation and storage.

A buffering agent may be used to maintain pH of the liquid compositionwithin an acceptable range for stability of IL-2. The buffering agentmay be an acid such as e.g., succinic acid, citric acid, phosphoricacid, and glutamic acid.

Examples of suitable bulking agents include e.g., glycine, mannitol, orvaline, or any combination thereof.

Examples of inert carriers which may be used as stabilizing agentsinclude sugars (e.g., sucrose, glucose, dextrose) and sugar alcohols, aswell as amino acids.

The pharmaceutical composition may additionally incorporate otherstabilizing agents, such as methionine, a nonionic surfactant such aspolysorbate 80, etc.

Specific examples of IL-2 formulations are described [10] and in U.S.Pat. No. 4,604,377.

Where IL-2 is in monomeric form, it is preferred to add to thecompositions an amino acid base sufficient to decrease aggregation ofIL-2 during storage. The amino acid base can be an amino acid or acombination of amino acids, where any given amino acid is present eitherin its free base form or in its salt form. Examples of such amino acidsinclude arginine, lysine, and aspartic acid.

In a particular embodiment, the composition comprises a multimeric IL-2,for example lyophilized.

A specific example of such a composition is Proleukin® IL-2. Thislyophilized formulation comprises selectively oxidized, recombinant IL-2admixed with a water soluble carrier, such as mannitol, that providesbulk, and SDS to ensure solubility of IL-2 in water. This composition issuitable for reconstitution in aqueous solutions for parenteralinjection.

Packaging

The current packaging of IL-2 is vials containing 18 M IU of IL-2. Giventhe low dose to be used, packaging for dose of 0.01M IU, 0.02 M IU, 0.5MIU 0.1M IU, 0.2 M IU, 0.5M IU, 1M IU and 3, or 3.5M IU are preferablyprepared.

A specific object of this invention therefore also resides in apharmaceutical composition comprising a unit dose of 3 M IU IL-2, orless. The composition may be in a vial, capsule, syringue, etc.

Treatment of Autoimmune and Inflammatory Diseases

The invention may be used for the treatment of any condition associatedto or caused by an undesirable immune response and/or in which a Tregquantitative or qualitative defect has been described. Examples of suchdiseases include autoimmune diseases, inflammatory diseases (includingany human disease in which an inflammation process plays an importantrole), as well as immune-related diseases.

The treatment may be curative or preventive. In a particular embodiment,the treatment is curative, i.e., concerns a subject in whom the diseaseis declared, even at very early stages. In such patients, the treatmentaims at reducing or stopping disease progression and/or suppressingdisease symptoms or causes. The treatment may lead to completedisappearance of disease in the patient, as illustrated in the examples.

The treatment may be preventive, i.e., in patients not having declared adisease or in patients in remission of a disease to prevent relapses,such as in the case of multiple sclerosis. In such patients, thetreatment aims at maintaining an elevated level of Tregs and/or reducinginflammation in order to avoid development of a disease caused byundesirable immune reaction.

Treatment designates any improvement in the patient condition, such as areduced pain, a reduced tissue injury, etc.

A Treg defect has been reported in various autoimmune human diseases,and the invention is particularly suited for treating inflammatory orautoimmune diseases, including without limitation HCV-relatedvasculitis, uveitis, myositis, type I diabetes, systemic lupuserythematous, systemic vasculitis, psoriasis, allergy, asthma, Crohn'sdisease, multiple sclerosis, rheumathoid arthritis, atherosclerosis,autoimmune thyroid disease, auto-inflammatory diseases,neuro-degenerative diseases, Alzeihmer disease, graft-versus-hostdisease, spontaneous abortion and allograft rejection. The invention maybe used as an adjunct treatment for inflammatory infection-relatedpathologies such as Chagas disease, Leishmaniasis, Helicobacter pyloriinfection, chronic viral hepatitis (B, C, D), HIV, HTLV, Malaria,Amoebiasis, or Shigellosis, all characterized by a high degree ofinflammation or conditions involving an inflammtory component affectingthe therapy such as post-surgical local inflammation.

Chronic HCV infection is uniquely associated with an array ofextrahepatic complications, which pathogenic mechanisms appear to belargely immunologically driven. Among these, cryoglobulinaemia and itsclinical sequelae hold the strongest association. Cryoglobulins arereadily detectable in 40-60% of HCV-infected patients [11, 20, 21, 22]whereas overt cryoglobulinaemia vasculitis (mixed cryoglobulinaemia: MC)develops in only 5-10% of the cases [11]. The most frequent targetorgans are skin, joints, nerves and kidney. The disease expression isvariable, ranging from mild clinical symptoms (purpura, arthralgia) tofulminant life-threatening complications (glomerulonephritis, widespreadvasculitis). The observation of T cells in vascular infiltrates, thepresence of autoantibodies and the observation that some HLA groupsconfer susceptibility to MC in HCV-infected patients support theautoimmune nature of this virus-linked pathology [12, 13]. MCpathophysiology appears to result from the interaction between HCV andlymphocytes, which directly modulates B- and T-cell function and resultsin polyclonal activation and expansion of B cell producing IgM withrheumatoid factor (RF) activity [23]. We previously reported that Tregsare significantly reduced in HCV-MC patients [12, 14, 15]. Moreover, inMC patients that could be succesfully treated for HCV, virus clearancewas associated vasculitis cure and Treg recovery.

The experiental section reports the results of a phase I/IIa studydesigned to assess the safety, immunological effects and clinicalefficacy of repeated administration of low-dose IL-2 therapy inHCV-infected patients with associated autoimmunity. We show thatlow-dose IL-2 is well tolerated, induces a dramatic and selectiveincrease in Treg cells, and leads to clinical improvement in allpatients treated. This is the first demonstration of in vivo Treginduction and recovery after IL-2 immunotherapy in a human autoimmunedisease.

As reported in the examples, the treatment was well tolerated and didnot induce effector T cells activation, vasculitis flare or increasedHCV viremia. Low-dose IL-2 dramatically increased the proportions ofCD4+CD25hiCD127−Foxp3+ Tregs with potent supressive activity in allpatients, and concommitantly decreased marginal zone B cellsproportions. Transcriptome studies of peripheral blood mononuclear cellsshowed that IL-2 induced a global atenuation of inflammatory/oxidativestress mediators. Reduced cryoglobulinemia and/or clinical improvementof the vasculitis were observed in 90% (9/10) and 80% (8/10) ofpatients, respectively.

This is the first demonstration of Treg cell recovery and clinicalimprovement after a well tolerated low-dose IL-2 immunotherapy in ahuman autoimmune disease. It paves the way for a broader use of IL-2 fortreating inflammatory and autoimmune diseases.

Other diseases include:

-   -   Systemic inflammatory response syndrome (SIRS) (choc)        (adjuvant);    -   Liver graft—neoadjuvant and adjuvant therapy for prevention of        inflammatory hepatic fibrosis—treating donor and/or recipient        for improved graft control    -   Liver cirrhosis—for prevention of inflammatory hepatic fibrosis        and related complications    -   Auto-inflammatory systemic diseases—reduction or prevention of        inflammatory reaction with clinical/biological flares    -   Acute respiratory distress syndrome (ARDS) (adjuvant)    -   Acute pancreatitis—reduction of inflammatory reaction and        secondary necrosis    -   Vascular surgery—stent—prevention of stent occlusion by        preventing inflammatory reaction to stent insertion    -   Improvement of cardiac remodelling following cardiac infarction    -   Orthopaedic surgery—reduction of inflammatory reaction following        OS    -   Odontology—treatment of parodonditis    -   Still disease    -   Psychiatry: Depression

The following examples are given for purposes of illustration and not byway of limitation.

EXAMPLES Example 1: Low-dose IL-2 in HCV-related Vasculitis

Here, we provide the first biological evidence in human subjects thatIL-2 can be used under conditions that induce Tregs without inducingTeffs in patients with auto-immunity. We report here the firstdemonstration of in vivo expansion of very potent supressive Tregthrough IL-2 immunotherapy in a human autoimmune disease, leading toclinical improvement. The primary end point of our study, increased Tregat the end of IL-2 therapy, and all secondary end points, including theclinical response were all met. We show that low-dose IL-2 is welltolerated, induces a dramatic and selective increase in Treg cells, andleads to clinical improvement in 80% of the patients. This is the firstdemonstration of in vivo Tregs induction and recovery after IL-2immunotherapy in a human autoimmune disease. Furthermore, we show forthe first time a marked anti-inflamatory effect of low-dose IL-2 inhumans.

METHODS Patients

Inclusion criteria for the study were as follows: 1) chronic active HCVinfection defined by a positive HCV RNA; 2) a history of MC vasculitisdefined by the (i) presence of serum cryoglobulin≧0.05 g/1 in at leasttwo determinations and (ii) the presence of the triadpurpura-arthralgia-asthenia or (iii) biopsy proven vasculitis (kidney,nerve or skin) in the absence of purpura [16, 17]; 3) presence atinclusion of a clinically active vasculitis with resistance orintolerance to conventional therapies, i.e. antiviral therapy(Peg-Interferon-α and ribavirin) and/or Rituximab; 5) a minimum of 6 or2 months after discontinuing Rituximab or antiviral therapy,respectively.

Exclusion criteria included co-infection with hepatitis B virus or HIV,liver cirrhosis, cancer or lymphoma, any immunosuppressant use in thelast 6 months, drug addiction, alcohol abuse or pregnancy.

Study Design

We conducted a monocenter open prospective phase I/II trial. Four cyclesof 5-days subcutaneous IL-2 therapy were administered. The first curewas carried out at the dose of 1.5 millions IU/day during a one weekhospitalization to evaluate tolerance. Based on satisfactorylytolerance, the 3 later cures were done ambulatory at the dose of 3millions IU/day. The second cure was started after a 10-day washout,while the following 2 cures after a 17-day washout period. The study wasapproved by institutional ethics committee and informed consent wasobtained from all patients.

Patients were evaluated on day-1 and day-5 of each cure, prior to thefirst and last IL-2 administration of that cure. They were alsoevaluated 48 to 90 days after the last IL-2 administration.

The response to treatment was analysed by comparing clinical,immunologic, and virologic parameters at the initial evaluation, at theend of each course of IL-2 (at weeks 1, 3, 6 and 9) and at the end offollow-up. Clinical response was defined by analysing the progression ofthe following main clinical signs: skin involvement (absence of purpuraand/or leg ulcer), peripheral neuropathy (clinical andelectrophysiologic improvement on two successive examinations), renalinvolvement (normalization of serum creatinine level and disappearanceof proteinuria and/or hematuria), and absence of arthralgia.

The primary end point was a 4% absolute increase of CD4⁺CD25⁺Foxp3⁺regulatory T cell (Treg) proportion at the end of IL-2 therapy (i.e. atW9). Secondary end points included safety, evaluation of celullar andhumoral immunity at W9, persistent increase of Treg levels at distancefrom treatment (W19) and evaluation of clinical response of thevasculitis.

Flow Cytometric Analysis

The immunomonitoring was performed according to previously publishedroutine methods used in the Pitié-Salpêtrière Biotherapy department.

Briefly, peripheral blood mononuclear cell (PBMC) subsets (CD3⁺, CD4⁺,CD8⁺ T lymphocytes, CD19⁺ B lymphocytes and CD3⁻CD56⁺ NK cell) counts(cells/μl) were established from fresh blood samples using CYTO-STATtetraCHROME kits with Flowcount fluorescents beads as internal standardand tetra CXP software with a FC500 cytometer according tomanufacturer's instructions (Beckman Coulter, Villepinte, France).Subsets of these cells were analysed using multicolour flow cytometryand mAbs directly conjugated to various fluorescent markers. Cellsacquisition and analysis by flow cytometry were performed using a FC500Cytometer (Beckman Coulter). Instrument setting parameters (gains,compensations, and threshold) were set with machine software (CXPSoftware; Beckman Coulter) in conjunction with calibration beads(Flow-set beads, Cytocomp kit, and CYTO-TROL Control Cells). Machinereproducibility was verified with standardized beads (Flow-check). Datawere analyzed with CXP analysis software (Beckman Coulter).

Subsets of these cells were analysed using multicolour flow cytometryand mAbs directly conjugated either to Fluorescein isothiocyanate(FITC), Phycoerythrin (PE), Phycoerythrin-Texas Red (ECD),Allophycocyanin (APC) or phycoerythrin-Cyanyn 7 (PE-Cya7) were used for:CD3-ECD or -PC7, CD4-ECD or -PC7, CD8-PC7, CD8-APC, CD14-PE, CD16-FITC,CD19-ECD, CD28-FITC, CD45RA-APC, CD45RO-FITC, CD56-PE CD69-PE, CD152-PEand HLA-DR-PC7 all from Beckman Coulter (Villepinte, France). CD25-PE,CD25APC, CD27-PE, CD62L-FITC and IgD-FITC, were from BD Biosciences (LePont De Claix, France). CD127 was from e-Biosciences (San Diego, Calif.,USA), and glucocorticoid-induced tumour necrosis factor-related protein(GITR)-PE from Miltenyi Biothech (Paris, France). Latency-associatedpeptide (LAP)-PE and CCR7-PE antibodies were from R&D Systems (Abingdon,UK). Intracellular CD152 labelling was performed using Fix and Permreagent from Invitrogen (Cergy Pontoise, France) after after CD3, CD4,CD127 and CD25 membrane staining. Matched mouse isotype controlantibodies were used. Intranuclear FOXP3 labeling was performed afterCD3, CD4, CD127 and CD25 membrane staining using APC anti-human Foxp3kit (PCH101 clone, eBiosciences) according to manufacturer'sinstructions. Rat IGg2a APC was used as isotypic control(eBiosciences,).

Cells acquisition and analysis by flow cytometry were performed using aFC500 Cytometer (Beckman Coulter). Instrument setting parameters (gains,compensations, and threshold) were set with machine software (CXPSoftware; Beckman Coulter) in conjunction with calibration beads(Flow-set beads, Cytocomp kit, and CYTO-TROL Control Cells). Machinereproducibility was verified with standardized beads (Flow-check). Datawere analyzed with CXP analysis software and Kaluza software (BeckmanCoulter)

For detection of intracellular cytokine production, PBMC were stimulatedwith 50 ng/ml PMA and 1 mM ionomycin in the presence of Golgi-Stop (BDBiosciences) for 4 hr and then stained with anti-IFN-g-FITC(eBioscience), or anti-IL-17-Alexa Fluor 647 (e-Bioscience) afterfixation and permeabilization, according to the manufacturer'sinstructions.

Suppression T Cell Assays

Cells suppression assay was performed as previously described. Briefly,PBMC were stained with appropriate combinations of mAbs in order topurify by flow cytometer (FACS Aria, BD Biosciences)CD3+CD4+CD25+CD127low/− cells namely FACS-sorted Treg. To test theirsuppressive activity, Treg were assayed in round-bottomed 96-well tissueculture plates mixed at various cell ratios (1/1; 1/2; 1/4; 1/8; 1/16)with 5×10⁴ autologous Facs-sorted CD4+CD25− cells as responder cells inthe presence of 10⁴ irradiated (15 grays) allogeneic PBMC as stimulatorcells in 200 μL of complete culture medium. Triplicates were performedfor each culture condition. After 4 days, cell proliferation wasdetermined by incorporation of 1 μCi (0.037 MBq) of ³H-thymidine(Amersham, Buckinghamshire, UK) for an additional 16 h and measuredusing a β-counter (counter-WALLAC). Results were expressed in counts perminute (cpm) and percentage of suppression of proliferation wasdetermined by proliferation of effector cells without treg/proliferationof effector cells with treg ratio.

Transcriptome Studies

RNAs were generated using RNeasy Mini Kit (QIAGEN, requis, CA) accordingto the manufacturer's instructions. RNA integrity was assessed using anAgilent Bioanalyser showing a quality of RNA integrity number of 7-9.5(Agilent Technologies). RNA yield was assessed using a NanoDrop 1000spectrophotometer (NanoDrop Products, Thermo Fisher Scientific).

Total RNA was amplified and converted to biotinylated cRNA according tothe manufacturer's protocol (Illumina TotalPrep RNA Amplification Kit;Ambion).

Labelled cRNA were hybridized overnight to Illumina Human HT-12 V3BeadChip arrays (Illumina), which contained more than 48,000 probes. Thearrays were then washed, blocked, stained and scanned on an IlluminaBeadStation following the manufacturer's protocols. Illumina BeadStudiosoftware (Illumina) was used to generate signal intensity values fromthe scans.

Data were normalized according to the quantiles method and supervisedhierarchical clustering was performed on 435 selected transcripts thatwere significantly modulated after treatment in at least 3 out of 6patients. Modulation threshold ratios were set up at 0.6 and 1.5 fordown and up-regulated transcripts respectively. One patient presentingtoo divergent modulation profiles from the others was excluded from theclustering analysis. Gene signatures were analyzed to generatefunctional networks using the PredictSearch™ software (Prediguard), adatamining bioinformatic solution dedicated to identify relevantcorrelations between genes and concepts, was used to generate functionalnetworks. This tool is daily updated with the whole NCBI Pubmed databaseand seeks for relevant correlations between gene-gene or gene-conceptwithin abstracts cocitations.

Correlations are also retrieved from the crosscomparison to the wholeset (>18000) of transcriptional signatures deposited in NCBI GEOdatabase and extracted with the DBF-MCL algorithm usingTranscriptomeBrowser tool. Furthermore PredictSearch™ allows theannotation of modulated genes according to KEGG and Biocarta pathways aswell as to GO ontology.

For unsupervised analyses, potential molecular signatures were extractedusing unsupervised Independent Component Analysis (ICA). Thosesignatures were added to the Gene Set Enrichment Analysis (GSEA)signature database and tested for their significance on our microarraydata using GSEA software. GSEA leading edges of statisticallysignificant molecular signatures (FDR q.value<0.05) were annotated fortheir GO terms and KEGG pathways enrichment. Signatures with enrichmentfor GO terms related to inflammation, immune response and autoimmunepathologies were selected. A Khi2 test was used to determine whetherthese selected signatures were preferentially up- or down-regulated inIL-2-treated samples as compared to the overall distribution of up- ordown-regulated signatures.

Statistical Analyses

A sample size of 10 patients achieves 94% power to detect a mean ofpaired differences of 4% with an estimated standard deviation ofdifferences of 3% and with a significance level (alpha) of 0.05 using atwo-sided Wilcoxon signed rank test, assuming that the actualdistribution of the percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cell isnormal.

Comparisons of baseline with W9 or Post-IL-2 measures were done with theWilcoxon signed rank test. The F approximation of the Friedman test wasused to compare across all repeated measurements [24]. Approximations ofthe critical region of the Friedman statistic and multiple comparisonswere performed where appropriate [25].

Time-to-peak values (Tmax) were determined directly from theexperimental data as the time of maximum Treg percentage (Emax).

RESULTS Patients

Ten patients were included (Table-1). At inclusion, the median (Q1-Q3)age was 58.5 (49.5-66.2) years with a 50/50% male to female ratio.Clinical manifestations of MC vasculitis included peripheral neuropathy(n=8), purpura (n=8), asthenia (n=6), arthralgia (n=3) and kidneyinvolvement (n=1) [daily proteinuria 1.5 g, microscopic hematuria andcreatininemia 74 μmol/L]. The median (Q1-Q3) cryoglobulin level was 0.53(0.26-2.77) g/L, of type II IgM Kappa in all cases. The median (Q1-Q3)C4 complement factor level was 0.065 (0.02-0.16) mg/L, rheumatoid factoractivity was present in 90% of cases and antinuclear antibodies werepositive in one patient (at a 1/640 titer). The median HCV viral loadwas 6.25 (5.5-6.8) Log copies/mL; no patients had liver cirrhosis.

Safety of Low Dose IL-2

Compliance to treatment was good and all patients completed all 4courses of IL-2. IL-2 was clinically and biologically well tolerated(Table-1). No significant changes in granulocytes, red blood cells, orliver enzymes were observed throughout the study. Only minor clinicalgrade 1 side effects were noted and spontaneously resolutive, includingasthenia (n=4), transient local reactions at injection sites (n=4),flu-like syndrome (n=4), myalgia (n=1) and hypertension (n=1). Notably,none of these occurred at the lowest, 1.5 million IU/day, IL-2 dosage(Table-1). During the entire treatment and follow-up, there were nobiological or clinical signs indicating activation of pathogenic Tcells: no vasculitis flare was noted; lymphoid organ examination did notshow abnormality suggestive of lymphoproliferative disorder induction;no increase in HCV viral load was observed (Table-1 & FIG. 1A).

Biological and Clinical Efficacy of Low Dose IL-2 on HCV-MC

The HCV viral load continuously decreased during the IL-2 treatmentperiod and was significantly lower at W9 (p=0.02), in the absence of anyantiviral treatment. Cryoglobulin serum levels, which also continuouslydecreased (P=0.014, at W9), were already decreased (P=0.003) while C4inversely increased (P=0.027) after the first 1.5 m IU IL-2 course (FIG.1). No patient developed antinuclear antibodies over IL-2 therapy andantinuclear antibodies diseappeared in patient #1.

In agreement with the improved biological parameters of HCV-MCvasculitis (FIG. 1), 8 out of 10 patients showed marked clinicalimprovement following IL-2 therapy, with disappearance of purpura andarthralgia (8/8 and 3/3 patients, respectively). Kidney parametersnormalized (daily proteinuria <0.3 g and absence of hematuria) inpatient #10. The only patients who did not have a marked clinicalresponse were those who presented with only a neuropathy at the time ofinclusion (n=2).

Low Dose IL-2 Induces a Dramatic Increase of CD4⁺ and CD8⁺ Regulatory TCells

IL-2 induced a dramatic increase in circulatingCD4⁺CD25^(hi)CD127⁻Foxp3⁺ Tregs (FIG. 2A and FIG. 3). The baselinepercentage of Tregs in this group of patients was of 3.6%±0.23(mean+/−sem), significantly lower than normal values (4.6±0.6) inagreement with our previously reported studies. At W9, Treg proportionswere of 11.8%±1.96 (P=0.004), reaching our primary efficacy criteriaendpoint. Noteworthy, Treg proportions were already increasedapproximately 2-fold after the first 5-day course of 1.5 million IU ofIL-2 (FIG. 2A). Treg proportions continued to increase during thewash-out period between courses, and were further boosted by thesubsequent courses (FIG. 2A). Compared to baseline values, theseincreased Treg proportions were statistically significant throughouttreatment (P=0.016 at W1 and P<0.001 at W3, 6 and 9). The median peakvalue of Tregs proportion (Emax=14%) was reached at the end of the thirdIL-2 course (median Temax=2.9), corresponding to an increase of 10.3%compared to baseline (Emax minus Baseline) or corresponding to a 350%increase of Tregs (Emax/Baseline). At distance (129 to 150 days) fromthe treatment, Treg proportion remained significantly elevated, at twicethe baseline value (6.1%±0.51, P=0.008), in the range of values fornormal blood donors. Finally, we evaluated the functionality of the theIL-2 expanded Tregs and found that they were highly suppressive (FIG.4).

A population of rare CD8⁺CD25^(hi)Foxp3⁺ T cells with supressor functioncan be detected in normal individuals |26] (FIG. 5). We monitored thesecells during the IL-2 treatment and observed their marked increase,concomitant to the CD4⁺ Treg increase. Following the first course theirproportion increased by approximately 500% and it remained elevatedthroughout treatment (FIG. 2A, Table-2). Altogether, the increase ofCD4⁺ and CD8⁺ Tregs leads to a striking increase in the global Treg/Teffratio, with no modification of the CD4/CD8 ratio (FIG. 2B, Table-2).

Total B cell numbers decreased during IL-2 treatment, immediately afterthe first course, and then recovered at distance from therapy. Thisdecrease particularly affected the IgD⁺CD27⁺ marginal zone B cells thatare implicated in the vasculitis pathophysiology [27] (FIG. 2C, FIG. 6).In contrast, there was a significant increase in numbers of NK cells,which was already detected following the first IL-2 course, and showed aclear tendency to continuous increase over time; this rapidly ceasedafter IL-2 discontinuation. There was notably a specific increase of theCD56^(bright) NK cells that produce high levels of immunoregulatorycytokines and are poorly cytotoxic (FIG. 2D, Table-2).

Low Dose IL-2 Induces a Global Decrease of Inflammation Revealed byTranscriptome Analyses of PBMCs

We analysed the transcriptome of PBMCs before and after IL-2 course(FIG. 3. We first performed a supervised analysis, comparing directlythe two sets of data and looking for genes that are up- ordown-regulated between the two conditions. This study first confirmedthe phenotypic observations, showing increased expression of genesrelated to Treg and NK cell function, together with a decreasedexpression of genes related to B cell function (not shown). Hierarchicalclustering and data mining approaches revealed a striking decrease inthe expression of genes associated with inflammatory/oxidative stressmediators (FIG. 7A). The NFKB pathway appears crucially involved in thisregulation (FIG. 7B). We confirmed these results by an unsupervised(i.e. non hypothesis driven) analysis. In this approach, all the pre-and post-IL-2 transcriptome data are mixed together, and signaturesmaximizing the segregation of the data in independent groups are soughtbased on Independent Component Analysis (ICA) [28]. Gene Ontology (GO)terms and pathways (GOTP) are then looked for among the signatures thatdifferentiated pre- and post-treated groups. The ratio of up-versusdown-regulated GOTP was of 0/251 for Inflammation (p=1,3E-40), 16/684for Immune responses (p=3,4E-94) and 77/555 for Lymphocyte Activation(p=7,0E-49)(FIG. 3C). Conversely, we obtained 1701 up- and 208down-regulated signatures enriched with the GOTP related to Cell Cycle(p=1,5E-138). Similar analysis with randomly chosen control terms showedno enrichment.

A similar analysis was then performed on Kyoto Encyclopedia of Genes andGenomes (KEGG) pathways terms related to autoimmune, inflammatory, andtransplantation-related pathologies and infectious diseases. Thesesignatures were preferentially down-regulated after IL-2 treatment(p=7.6E-09 and p=7.6E-36, respectively), while control pathologies werenot.

TABLE 1 Characteristics and outcome of HCV related autoimmune vasculitispatient under IL-2 therapy Characteristics Patient 1 Patient 2 Patient 3Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10Age at diagnosis (years) 48 74 63 50 59 67 51 66 58 43 Gender femalefemale male male male male female male female female Symptoms BaselineP, A, F P, N, F P, N, F N, F P, N, F P, N P, N N A, P A, P, N, K, F PostIL-2 therapy — — N N — — — N — N Previous therapy Peg Peg Peg Peg PegPeg Peg Peg Peg Peg IFN/RBV IFN/RBV IFN/RBV IFN/RBV IFN/RBV IFN/RBVIFN/RBV IFN/RBV IFN/RBV IFN/RBV Rituxan Rituxan Cryoglobulin serum level(g/L) Baseline 0.56 1.61 0.17 0.16 0.30 0.3 6.99 2.77 2.78 0.51 PostIL-2 therapy 0 1.00 0.91 0 0.34 0 3.87 1.99 2.94 0.19 C4 complementlevel (g/L) Baseline 0.20 0.06 0.08 0.20 0.06 0.15 0.07 0.02 0.03 0.02Post IL-2 therapy 0.21 0.05 0.12 0.27 0.09 0.19 0.10 0.06 0.04 0.03 HCVviral load (Log UI/mL) Baseline 5.3 6.2 7.2 6.3 5.6 6.9 5.8 6.3 6.8 5.4Post IL-2 therapy 5.2 5.8 5.6 6.2 5.6 5.8 6.0 5.1 4.6 5.5 Alanineaminotransferase (IU/L) Baseline 26 38 29 25 77 40 58 60 104 40 PostIL-2 therapy 19 17 45 24 81 23 22 62 80 35 IL-2 therapy Side effectsCourse 1 (1.5 M U/day for — — — — — — — — — — 5 days) Course 2 (3 MU/day for — F, M F F F Flu, LR Flu, LR Flu, LR AH Flu, LR 5 days) Course3 (3 M U/day for — F F F F LR Flu Flu, LR AH — 5 days) Course 4 (3 MU/day for — — — — — LR — LR — — 5 days) A: arthralgia, F: fatigue, P:purpura, N: neuropathy, K: kidney involvement, M: myalgia, Flu: Flu-likesyndrom, LR: local reaction, AH: arterial hypertension

TABLE 2 Immunologic characteristics under IL-2 therapy Baseline End ofCourse 1 End of Course 2 End of Course 3 End of Course 4 Post IL-2Lymphocytes (cells/μl) 685 838 746 781 855 871 (242-2008) (267-1817)(238-1529) (398-2320) (558-1859) (426-2302) T cells (cells/μl) 633 655 456* 506 647 690 (242-1214) (401-1214) (284-1334) (250-1237) (292-1660)(299-936) Total CD4 T cells (cells/μl) 298 456 335 403 340 531(149-1193) (118-1009) (91-658)  (94-1192) (173-1041) (151-984)  TotalCD8 T cells (cells/μl) 234 191  130*  147* 223 292 (73-681) (36-531)(64-414) (77-680) (104-448)  (84-518) CD4/CD8 ratio    1.9    1.8    1.6   1.7    1.96    1.8 (1.4-4.3)   (1-5.3) (1.1-5.2)  (0.9-5.1) (0.8-4.9)  (1.3-4.9)  NK cells (cells/μl)  63  97*  155**  164**  209***  77 (49-184) (44-245) (13-290) (50-269) (79-389) (41-229) CD56bright NK cells (cells/μl)  8   18**   27***   20**   27**    7***(5-17) (8-30)  (2-100) (6-58) (8-93) (2-12) B cells (cells/μl) 118  90 84*  86*  98 111 (18-570) (20-334) (27-236) (24-342) (29-175) (30-522)MZ B cells (cells/μl)  12   4*   3*   3**   4**  3 (1-77) (1-35) (1-14)(1-15) (1-14) (1-42) Treg CD4+CD25hiCD127-  15  25   29***   31**   36**  31** Foxp3+ (cells/μl) (5-29) (8-71) (9-92) (14-221) (20-271) (12-62) % CD25hiCD127-FoxP3+/CD4+    3.8    6.8*     10.4***   10***   11***  6** (1.8-4.4)  (3.7-15)   (5-24) (3.2-29)   (6-26) (3-8)  CD8+Foxp3+(cells/μl)    0.08     0.46*     0.46*     0.62**     0.47**    0.19(0.03-1.36)  (0.03-3.71)  (0.11-3.31)  (0.12-6.8)  (0.17-2.68) (0.07-0.64)  % CD25+FoxP3+/CD8+    0.04      0.35***      0.24***    0.2***      0.28***    0.08 (0.01-0.2)  (0.02-0.7)  (0.08-0.99) (0.11-1)    (0.08-0.6)  (0.03-0.24)  % Treg/Teff (CD4+CD8)  4     9.5**  13***     14.5***   14***   6** (2-6)  (4-17) (9-53) (6-42) (7-32)(4-13) CD4+ T cells % CD25+ (without Treg)  44  29   16***   23***  28**  46 (20-59)  (10-63)  (5-32) (10-40)  (7-48) (17-66)  Resultsshown are median with range in parentheses for each cell populationsanalysed for the 10 patients included in the protocol. Statisticalanalysis was done by Wilcoxon ranked-sum test; *P < 0.05; **P < 0.01;***P < 0.001 versus baseline

Example 2: Low-dose IL-2 in Type 1 Diabetes

The inventors initiated an IL-2 dose-finding clinical trial in T1D,which aimed to define the lowest active dose that could safely induceTregs in adult T1D patients.

The DF-IL2 trial is double blinded, comparing placebo, 0.3, 1 and 3mIU/day Proleukin® doses (cumulative dose of 1.5, 5 and 15 mIU,respectively).

The objective of the trial will be to preserve remaining endogenousinsulin secretion in patients with recently diagnosed T1D.

Main patient characteristics: adults, both sexes, T1D diagnosis asWHO-ADA, disease duration since diagnosis of less than 12 weeks at firstIL-2 dosing and detectable C-peptide at entry.

The current recommendation for a clinically meaningful effect is totarget with the active treatment at least a preservation of pancreaticβ-cell mass, i.e. the maintenance of C-peptide AUCO-120 compared tobaseline.

All 24 patients have been included. Although the investigators are stillblind to the IL-2 dose administered, the results obtained so far showthat IL-2 is safe (no SAE in any of the patients). Minor adverse events(mild fever, moderate pain at the injection site . . . ) have beenobserved in some patients and based on safety data from our previousclinical trial40 are most likely related to the dose of 3 mIU/day. Aninterim analysis of the Treg dynamic for the first 16 patients who havecompleted their treatment has been performed by an independentstatistician and without un-blinding the study to the investigators(following authorisation from regulatory authorities). This interimstudy shows that (i) IL-2 can indeed induce Tregs in T1D patients and(ii) the dose of 1 mIU per day induces a good Treg response that lastover 2 weeks (see FIG. 8).

Importantly, it was not detected in patients significant decrease ofC-peptide production. C-peptide measurement after a MMTT (mixed-mealtolerance test) shows at least preserved or sometimes increasedC-peptide production 2 months after the 5-day IL-2 treatment inpatients, as shown for patient 1 on FIG. 9B (note that we do not knowthe dose received by this patient, but that the increase in Tregobserved during treatment indicates that he received IL-2, not theplacebo).

Following this first study with adult T1D patients, the inventorsconceived a second dose finding trial, for childhood T1D, with inclusionof patients from ≧7 years old (DF-IL2-child). We refined our dosefinding study based on our results in the above-described trial. A doserange of 0.125 to 1 mUI/m2/d (here defined as ultra-low dose) is beinginvestigated over a one-year treatment period (16 injections), with theaim to confirm tolerance and efficacy for Treg induction. Thiscorresponds to a cumulative dose of 2 to 16 mUI/m2, more than 100 timesless than in the above described study.

Collectively, our clinical results relieve the concern that IL-2 mightnot be as effective in Treg induction in T1D patients who havealterations of the IL-2/IL-2R activation pathway. They also confirm theexcellent tolerance of IL-2 at the doses used and support that 0.5mUI/m2/day is an appropriate dose for assessing IL-2 efficacy inrecently diagnosed T1D patients, children and adults.

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The invention claimed is:
 1. A method for stimulating regulatory Tlymphocytes (Tregs) in a human subject suffering from type T diabetes,the method comprising subcutaneously administering interleukin-2 (IL-2)to the human subject at a dose of between 0.1 and 3.5 MIU/day in a firstcourse of treatment, wherein the first course of treatment is followedby a maintenance dose, wherein the first course of treatment consists ofan administration of IL-2 once per day for at least 3 consecutive days,and wherein the maintenance dose is given to the subject once a week oronce or twice a month.
 2. The method of claim 1, wherein the humansubject is administered with IL-2 at a dose of 3 MIU/day in the firstcourse of treatment.
 3. The method of claim 1, wherein the human subjectis administered with IL-2 at a dose of less than 2 MIU/day in the firstcourse of treatment.
 4. The method of claim 1, wherein the subject isadministered with IL-2 at a dose of between 0.25 and 3.5 MIU/day in thefirst course of treatment.
 5. The method of claim 1, wherein the firstcourse of treatment consists of an administration of IL-2 once per dayfor 3 to 7 days.
 6. The method of claim 1, wherein the maintenance dosestarts 1 to 4 weeks after the last dose of the first course oftreatment.
 7. The method of claim 1, wherein the subject is administeredwith IL-2 at a dose of 1.5 MIU/day in the first course of treatment. 8.The method of claim 1, wherein the subject is a child.
 9. The method ofclaim 1, wherein the maintenance dose is given to the subject once aweek.
 10. The method of claim 1, wherein the maintenance dose is givento the subject once or twice a month.
 11. A method for stimulatingregulatory T lymphocytes (Tregs) in a human subject suffering from typeI diabetes, the method comprising subcutaneously administeringinterleukin-2 (IL-2) to the human subject at a dose of between 0.05 and2 MIU/m²/day in a first course of treatment, wherein the first course oftreatment is followed by a maintenance dose, wherein the first course oftreatment consists of an administration of IL-2 once per day for atleast 3 consecutive days, and wherein the maintenance dose is given tothe subject once a week or once or twice a month.
 12. The method ofclaim 11, wherein the subject is administered with IL-2 at a dose ofbetween 0.2 and 1 MIU/m²/day in the first course of treatment.
 13. Themethod of claim 11, wherein the human subject is administered with IL-2at a dose of 0.2 to 0.6 MIU/m²/day in the first course of treatment. 14.The method of claim 11, wherein the subject is a child.
 15. The methodof claim 11, wherein the first course of treatment consists of anadministration of IL-2 once per day for 3 to 7 days.
 16. The method ofclaim 15, wherein the maintenance dose starts 1 to 4 weeks after thelast dose of the first course of treatment.
 17. The method of claim 11,wherein the maintenance dose is given to the subject once a week. 18.The method of claim 11, wherein the maintenance dose is given to thesubject once or twice a month.